A medium combination and method for inducing plant regeneration through peony petiole

Abstract

The present application belongs to the field of plant tissue culture, and particularly relates to a culture medium combination and method for inducing plant regeneration through peony petiole, comprising: a callus induction tissue culture medium and an adventitious bud differentiation medium are both MS medium containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA; a rooting induction medium is 1 / 2MS medium containing 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA and 0.4-0.6 g / L TA. The peony aseptic test tube seedling petiole is used as an explant; the explant is inoculated into the above callus induction medium for dedifferentiation culture, and callus is obtained; the callus is inoculated into the above adventitious bud differentiation medium for bud differentiation induction, and adventitious buds are obtained; the adventitious buds are inoculated into the above rooting induction medium for rooting induction after subculturing, and regenerated seedlings are obtained. Through the combination of the culture medium and the tissue culture method of the present application, the peony seedling petiole is used for tissue culture, plant seedlings are obtained, the efficiency of obtaining tissue culture seedlings is high, the adventitious bud and adventitious root induction effects are good, and the transplanting survival rate is high.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture, specifically relating to a culture medium combination and method for inducing plant regeneration through peony petioles. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Peony, a traditional Chinese flower, is hailed as "the national flower of beauty and heavenly fragrance" and "the king of flowers." In recent years, as the oil, cut flower, and health benefits of peonies have been increasingly recognized, they have become an important economic plant combining ornamental, medicinal, and edible value. Their position in the flower industry is rising, and their market prospects are broad. Traditionally, peonies have been bred through hybridization, which is characterized by long breeding cycles, low efficiency, and slow propagation, severely hindering variety updates and limiting the high-quality development of the industry. Therefore, the lack of specialized varieties is a significant problem facing the development of the peony industry.

[0004] Transgenic technology can improve breeding efficiency, and a stable and efficient plant regeneration system is the foundation for cultivating new varieties using genetic engineering. There have been several reports of successfully cloning peony genes, and theoretically, peony varieties with specific needs can be cultivated through transgenic technology.

[0005] Currently, a complete regeneration system for peonies has not been established, preventing the application of transgenic technology in peony breeding. Domestic and international scholars have successively conducted research on peony plant regeneration systems using peony leaves, stem segments, cotyledons, receptacles, and seeds as explants, but a complete regeneration system has yet to be established. The main problems include: (1) severe browning of callus tissue, resulting in poor proliferation; (2) low adventitious bud differentiation rate; (3) difficulty in inducing adventitious roots; and (4) transplant failure. Therefore, establishing a complete peony plant regeneration system and subsequently conducting genetic transformation research is urgently needed. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a culture medium combination and method for inducing plant regeneration through peony petioles. The peony regeneration method provided by the present invention solves at least one of the aforementioned problems.

[0007] To achieve the above objectives, a first aspect of the present invention provides a culture medium combination for inducing plant regeneration through peony petioles, comprising a callus induction medium, an adventitious bud differentiation medium, and a rooting induction medium.

[0008] The callus induction culture medium and adventitious shoot differentiation culture medium were both MS medium containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA;

[0009] The rooting induction medium is a 1 / 2 MS medium containing 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA and 0.4-0.6 g / LTA.

[0010] In some embodiments, the rooting induction medium further includes Ca 2+ The preferred concentration is 0.003 mol / L.

[0011] In some embodiments, an adventitious bud proliferation medium is also included, said adventitious bud proliferation medium containing Ca 2 + MS medium containing 1.2-1.8 mg / L 6-BA and 0.03-0.07 mg / L NAA.

[0012] A second aspect of the present invention provides a method for inducing plant regeneration through peony petioles, comprising:

[0013] The petioles of sterile peony test-tube seedlings were used as explants;

[0014] The explants were inoculated into the above-mentioned callus induction medium for dedifferentiation culture to obtain callus tissue.

[0015] The callus tissue was inoculated into the above-mentioned adventitious bud differentiation medium to induce bud differentiation and obtain adventitious buds;

[0016] After dividing the adventitious buds, they were inoculated into the above-mentioned rooting induction medium to induce rooting and obtain regenerated seedlings.

[0017] In some embodiments, the adventitious shoots generated by induced differentiation are inoculated into the above-mentioned adventitious shoot proliferation medium for proliferation culture, and then induced to root.

[0018] In some embodiments, after the adventitious buds are divided, they are inoculated into the above-mentioned rooting induction medium, first subjected to dark treatment, and then cultured under light conditions; preferably, the dark treatment time is 5-9 days, more preferably 7 days.

[0019] In some implementations, the regenerated seedlings are induced to produce dormant buds, and then the dormancy of the dormant buds is broken, followed by hardening off and transplanting.

[0020] In some embodiments, the regenerated seedlings are inoculated into a dormant bud induction medium to produce dormant buds. The dormant bud induction medium includes 0–2.0 mg / L IBA, 0–0.1 mg / L NAA, and 50–200 mL / L coconut juice.

[0021] In some implementations, regenerated seedlings with dormant buds are cultured in the dark at 2-8°C to break the dormancy of the buds before transplanting.

[0022] In some implementations, the regenerated seedlings that have broken dormancy are transplanted, with Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus megaterium added to the transplanting substrate.

[0023] In some implementations, the transplanting substrate includes peat moss and perlite.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. The culture medium combination of the present invention is applied to the tissue culture regeneration system of peony aseptic seedling petioles. By combining the culture medium of the present invention with the tissue culture method, peony seedling petioles are used for tissue culture to obtain plant seedlings. The efficiency of obtaining tissue culture seedlings is high, the induction effect of callus, adventitious buds and adventitious roots is good, and the transplant survival rate is high. The callus induction medium can improve the callus induction ratio of explants and improve the quality of callus tissue. The adventitious bud differentiation medium can improve the differentiation rate of adventitious buds. The rooting induction medium further improves the rooting rate of tissue culture plants, greatly improving the differentiation rate of adventitious buds and the rooting rate.

[0026] 2. The present invention can break the dormancy of the terminal bud after the test-tube seedlings have taken root, and can make the root system develop well, grow normally after transplanting, and establish a complete plant regeneration system.

[0027] 3. This invention uses the petioles of peony seedlings for tissue culture to obtain plant seedlings. The effect of inducing callus tissue is better than that of other parts. The callus induction ratio of the explants is high and the quality of the callus tissue is high. Attached Figure Description

[0028] Figure 1 These are physical images of different stages of the peony regeneration technology of the present invention. Detailed Implementation

[0029] Given that existing techniques for peony tissue culture via explants mostly only induce callus tissue without differentiating adventitious buds, and even when a few adventitious buds are induced and rooted plants are obtained, the rooting rate is very low, and transplanting often fails. Therefore, the regeneration system is far from robust. This invention proposes a culture medium combination and method for inducing plant regeneration through peony petioles. This scheme establishes a complete callus regeneration system, laying the foundation for peony molecular biology research and the application of transgenic technology.

[0030] The following are the English abbreviations of the hormones involved in this invention: TDZ (thiafenuron, N-phenyl-N-1,2,3-thiadiazole-5-urea), NAA (naphthaleneacetic acid, 2-(1-naphthyl)acetic acid), 6-BA (6-benzylaminopurine), IBA (3-indolebutyric acid), TA (triacontanol), 2,4-D (2,4-dichlorophenoxyacetic acid), and ZT (zeatin, 6-trans-4-hydroxy-3-methyl-but-2-enylaminopurine).

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] It should be noted that the materials and raw materials used in the following examples are all commercially available.

[0033] Example

[0034] This invention uses the peony variety 'Hongxia Lanman' as an example to specifically describe the culture medium combination and method for inducing plant regeneration through peony petioles. It should be noted that other peony varieties can also achieve the same or similar results.

[0035] 1. Callus induction

[0036] 1.1 Effects of different parts of test-tube seedlings on callus induction

[0037] Preparation of callus induction medium: The callus induction medium was MS medium supplemented with hormones, sugar 30 g / L and agar 6 g / L as shown in Table 1;

[0038] Using the petioles and leaves of sterile peony test-tube seedlings as explants, the petioles and leaves were cut into small segments of about 0.5 cm on a clean bench.

[0039] Petiole and leaf segments were inoculated into different callus induction media as shown in Table 1 for culture. The culture conditions were: temperature (23±1)℃, light intensity 2000 lx, light duration 12 h·d⁻¹, and induction time 35 days. The callus induction effect was observed after 35 days.

[0040] As shown in Table 1, when inducing callus tissue using test-tube seedling leaves and petioles, the induction rate of petioles was better than that of leaves under different hormone combinations. Moreover, when using test-tube seedling leaves for induction, the leaves showed severe browning overall, and the induced callus was very small. The induction effect of petioles was better than that of leaves, and the effect was best under the hormone combinations of ZT+NAA and TDZ+NAA.

[0041] Table 1. Effects of different parts of in vitro plantlets on callus induction.

[0042]

[0043] 1.2 Different hormone combinations induce callus tissue

[0044] Preparation of callus induction medium: The callus induction medium was MS medium supplemented with hormones, sugar 30 g / L and agar 6 g / L as shown in Table 2;

[0045] Using the petioles of sterile peony test-tube seedlings as explants, the petioles were cut into small segments of about 0.5 cm on a clean bench.

[0046] Petiole segments were inoculated into different callus induction media as shown in Table 2 for culture. The culture conditions were: temperature (23±1)℃, light intensity 2000 lx, and photoperiod 12 h·d. -1 The induction time was 35 days. The induction and culture effect of callus tissue was observed after 35 days.

[0047] Table 2 shows that the callus color and volume of group 4 were better than those of groups 1-3. The optimal combination of hormones in the culture medium for inducing callus in petiole was TDZ and NAA, with a callus induction rate of 84.6%. The induced callus was light green and larger in size.

[0048] Table 2. Effects of different hormone combinations on callus induction.

[0049]

[0050] 1.3 Different concentrations of hormones induce callus tissue

[0051] Preparation of callus induction medium: The callus induction medium was MS medium supplemented with hormones, sugar 30 g / L and agar 6 g / L as shown in Table 3;

[0052] The treated petiole segments were inoculated into different callus induction media as shown in Table 3 and cultured under the same conditions. The callus induction culture effect was observed after 35 days.

[0053] Table 3 shows that the callus induction effect of groups 4-7 was better, with group 4 having the best effect and group 7 having the worst effect. The optimal hormone concentration was TDZ 1.0 mg / L + NAA 0.1 mg / L.

[0054] Table 3. Effects of different hormone concentrations on callus induction.

[0055]

[0056]

[0057] 2. Adventitious bud differentiation

[0058] 2.1 Different hormone combinations induce bud differentiation

[0059] Preparation of adventitious shoot differentiation medium: The differentiation medium was MS medium supplemented with hormones, sugar 30 g / L and agar 6 g / L as shown in Table 4;

[0060] The callus tissue was cut into two segments and cultured separately in the differentiation medium shown in Table 4 to induce shoot differentiation. Culture conditions: temperature (23±1)℃, light intensity 2000 lx, photoperiod 12 h·d. -1 The culture period is 40 days, and the effect is observed after 40 days.

[0061] As shown in Table 4, the number of induced callus and the bud differentiation induction rate of Group 3 were significantly better than those of Groups 1-2. The optimal medium for bud differentiation was MS + TDZ 1.0 mg / L + NAA 0.1 mg / L + sugar 30 g / L + agar 6 g / L, with a bud differentiation rate of 51%.

[0062] Table 4. Effects of different hormone concentration combinations on bud differentiation

[0063]

[0064] 2.2 Different hormone concentrations induce bud differentiation

[0065] Preparation of adventitious shoot differentiation medium: The differentiation medium was MS medium supplemented with hormones, sugar 30 g / L and agar 6 g / L as shown in Table 5;

[0066] The callus tissue was cut into two segments and cultured separately in the differentiation medium shown in Table 5 to induce shoot differentiation. The culture conditions were the same as above, and the results were observed after 40 days.

[0067] As shown in Table 5, groups 3-5 were significantly better than group 6, with group 3 showing the best results.

[0068] Table 5. Effects of different hormone concentrations on bud differentiation

[0069]

[0070] 3. Adventitious bud proliferation

[0071] Preparation of adventitious bud proliferation medium: The bud proliferation medium is MS medium supplemented with the hormones shown in Table 6, 150 mL / L coconut juice, 30 g / L sugar and 6 g / L agar;

[0072] Differentiated adventitious buds were cut and inoculated into the bud proliferation medium shown in Table 6 for proliferation culture, under the same conditions as above, for 45 days. The results were observed after 45 days.

[0073] As shown in Table 6, different combinations of hormone concentrations had varying effects on shoot proliferation, with proliferation coefficients ranging from 1.52 to 2.36. Groups 1-3 showed better results than group 4, with group 2 exhibiting the best shoot proliferation effect. The optimal culture medium formula for shoot proliferation was MS + Ca. 2+ The growth coefficient reached 2.36 when the solution was 0.003 mol / L + 6-BA 1.5 mg / L + NAA 0.05 mg / L + coconut juice 150 mL / L + sugar 30 g / L + agar 6 g / L, and the buds grew vigorously.

[0074] Table 6. Effects of different hormone concentration combinations on bud proliferation

[0075]

[0076]

[0077] 4. Rooting Induction

[0078] 4.1 Rooting induced by different concentrations of hormones

[0079] Preparation of rooting induction medium: The rooting induction medium is prepared by adding the hormones and Ca shown in Table 7. 2+ 1 / 2 MS medium containing 40 g / L sugar and 6 g / L agar;

[0080] Select the above-mentioned adventitious buds (i.e. rootless test-tube seedlings) with a height of 2cm or more, cut them into single plants, and inoculate them into the rooting medium shown in Table 7 to induce the occurrence of adventitious roots. The culture conditions are the same as above. After 45 days of culture, the rooting status is counted.

[0081] As shown in Table 7, groups 1-4 were all superior to group 5, with group 4 showing the best results, achieving a rooting rate of 70%, producing robust roots with minimal basal callus. Therefore, the optimal culture medium formula for inducing adventitious root formation is: 1 / 2 MS + Ca... 2+ 0.003 mol / L + IBA 3.0 mg / L + NAA 0.5 mg / L + TA 0.5 g / L + sugar 40 g / L + agar 6 g / L.

[0082] Table 7 Effects of different hormone concentration combinations on adventitious root induction

[0083]

[0084] 4.2 Effect of Dark Treatment Time on Adventitious Root Induction

[0085] Inoculate rootless test-tube seedlings

[0086] Ca 2+The effects of different dark treatment times on adventitious root induction were studied in 1 / 2 MS medium containing 0.003 mol / L IBA 3.0 mg / L NAA 0.5 mg / L TA 0.5 g / L sugar 40 g / L agar 6 g / L. The dark treatment times are shown in Table 8. After that, the medium was placed under light conditions and cultured under the same conditions. Rooting was recorded after 45 days of culture.

[0087] As shown in Table 8, different durations of darkness treatment can promote the formation of adventitious roots. The optimal treatment time is 7 days, with a rooting rate of 90.8%.

[0088] Table 8. Effects of different dark treatment times on adventitious root induction.

[0089] 3 77.8 The root system is robust, with little callus tissue at the base. 5 82.0 The root system is robust, with little callus tissue at the base. 7 90.8 The root system is robust, with little callus tissue at the base.

[0090] 5. Dormant bud induction

[0091] Prepare a dormant bud induction medium consisting of 1 / 2 MS medium containing 40 g / L sugar and 6 g / L agar. The induction period is approximately 60 days.

[0092] The tissue culture seedlings were transferred to a dormant bud induction medium for further culture under the following conditions: 15–20℃, light intensity 2000–3000 lx, and photoperiod 10–12 h·d. -1 A terminal bud forms after approximately 60 days of cultivation. This is existing technology; see patent CN 113875596 A for details.

[0093] 6. Hibernation ends

[0094] Test-tube seedlings that have developed dormant buds are placed in the dark at 4℃ to break dormancy. The culture period is 40-50 days. Once the dormant buds begin to sprout, they are removed, hardened off, and transplanted.

[0095] 7. Hardening off seedlings and transplanting

[0096] After the above-mentioned tissue culture seedlings that have been released from dormancy of the apical buds are hardened off to allow them to gradually adapt to the external environment, they are then transplanted:

[0097] During hardening, the tissue culture seedlings are hardened for 8 to 12 days under a gradient temperature environment (10 to 23°C) from low to high (the specific gradient design is, for example, increasing the temperature by 1 to 3°C per day, and culturing for 1 to 2 days for each temperature gradient).

[0098] When transplanting, the transplanting substrate is a mixture of peat moss and perlite in a volume ratio of 3:1;

[0099] To ensure a high survival rate, during the initial transplanting period (within 10-20 days after transplanting), the environmental conditions should be controlled as follows: relative humidity above 90% and temperature between 20-25℃. The survival rate can reach over 85%. Additionally, 0.2-0.4g / plant of a compound microbial agent (containing Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus megaterium) can be added. This compound microbial agent is Yangshao compound microbial agent purchased from Henan Yangshao Biochemical Engineering Co., Ltd.

[0100] This is to prevent stem rot in tissue culture seedlings and promote the development of fine roots. Table 9 shows the survival rate two months after transplanting. The results show that adding 0.2-0.6g of microbial agent per plant can effectively prevent stem rot and significantly improve the survival rate of test-tube seedlings. However, the difference between adding 0.2-0.6g is not significant. Considering cost, 0.2-0.4g per plant is recommended.

[0101] Table 9. Effects of different dosages of microbial inoculants on the survival rate of transplanted in vitro seedlings.

[0102]

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An application of a culture medium combination for culturing peony petioles to induce plant regeneration, characterized in that, Including callus induction medium, adventitious shoot differentiation medium, adventitious shoot proliferation medium and rooting induction medium; The callus induction medium was MS medium containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA. The adventitious bud differentiation medium was MS medium containing 0.8-1.2 mg / L TDZ and 0.08-0.12 mg / L NAA. The adventitious bud proliferation medium is: 0.003 mol / L Ca. 2+ MS medium containing 1.2-1.8 mg / L 6-BA and 0.03-0.07 mg / L NAA; The rooting induction medium consisted of 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA, 0.4-0.6 g / L TA, and 0.003 mol / L Ca. 2+ 1 / 2 MS medium.

2. A method for inducing plant regeneration through peony petioles, characterized in that, include: The petioles of sterile peony test-tube seedlings were used as explants; Explants were inoculated into callus induction medium for dedifferentiation culture to obtain callus tissue. Callus tissue was inoculated into adventitious bud differentiation medium to induce bud differentiation and obtain adventitious buds; The adventitious shoots generated by induced differentiation were inoculated into the adventitious shoot proliferation medium for proliferation culture, and then rooting was induced. After dividing the adventitious buds, they were inoculated into a rooting induction medium to induce rooting and obtain regenerated seedlings; The callus induction medium was MS medium containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA. The adventitious bud differentiation medium was MS medium containing 0.8-1.2 mg / L TDZ and 0.08-0.12 mg / L NAA. The adventitious bud proliferation medium is: 0.003 mol / L Ca. 2+ MS medium containing 1.2-1.8 mg / L 6-BA and 0.03-0.07 mg / L NAA; The rooting induction medium consisted of 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA, 0.4-0.6 g / L TA, and 0.003 mol / L Ca. 2+ 1 / 2 MS medium.

3. The method for inducing plant regeneration through peony petioles as described in claim 2, characterized in that, After dividing the adventitious buds, they were inoculated into rooting induction medium, first treated in the dark, and then cultured under light conditions.

4. The method for inducing plant regeneration through peony petioles as described in claim 3, characterized in that, The dark processing time is 3-7 days.

5. The method for inducing plant regeneration through peony petioles as described in claim 4, characterized in that, The dark processing time is 7 days.

6. The method for inducing plant regeneration through peony petioles as described in claim 2, characterized in that, Induce dormant buds into the regenerated seedlings, then break the dormancy of the buds and harden them off before transplanting.

7. The method for inducing plant regeneration through peony petioles as described in claim 2, characterized in that, The regenerated seedlings were inoculated into a dormant bud induction medium to induce dormant buds. The dormant bud induction medium consisted of 1 / 2 MS medium containing 2.0 mg / L IBA, 0.1 mg / L NAA, 50–200 mL / L coconut juice, 40 g / L sugar, and 6 g / L agar. The regenerated seedlings that produced dormant buds were then cultured in the dark at 2–8°C to break the dormancy of the buds before transplanting.

8. The method for inducing plant regeneration through peony petioles as described in claim 2, characterized in that, The regenerated seedlings that have broken dormancy are transplanted. The transplanting substrate includes peat moss and perlite, and also includes 0.2-0.6 g / plant of compound microbial agent, which contains Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus megaterium.

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